Antibiotic production



DSG 19, 1961 c. J. BEssL-:LL x-:TAL 3,013,947

ANTIBIOTIC PRODUCTION Filed June 14, 1960 3,013,947 ANTEBITHC PRODUC'HON Christine .loy Besseil, Stoke Pages, Winston Kenney Anslow, Slough, Aileen Marion Mortimer, Seliy Galt, Birmingham, Derek Lawrence Fletcher, Stoke Poges, and Alan Rhodes, Bracknell, England, assignors to Glaxo Laboratories Limited, Greenford, England, .a British company Filed .lune 14, 1960, Ser. No. 36,058 12 Claims. (Cl. 2195-80) This invention is concerned with the production of an important antifungal antibiotic which We have designated lagosin The antibiotic lagosin is believed to be identical with the antibiotic designated Pentamycin which is described in the Journal of Antibiotics, Series A, 1958, pages 26-29. Pentamycin is described as being produced by the culture of Strepllomyces penticus. For convenience the antibiotic with which this invention is concerned will continue herein to be termed lagosin We have found that lagosin may be obtained in advantageous manner by the aerobic cultivation of an organism newly discovered by us which we have identified as belonging to the genus Streptomyces. This organism, which is isolated from a soil sample taken in Nigeria has been designated Streptomyces roseo-luteus by which name it will be referred to hereinafter. Cultures of the organism Streptomyces roseo-luteI/.s have been deposited in the collection of the North Regional Research Laboratory, Peoria, illinois, United States of America, where it has been assigned the number NRRL 2776. It has also been deposited in the National Collection of Industrial Bacteria at the National Chemical Laboratory, Teddington, England, where it has been assigned the number N.C.I.B. 8984. The use of the new organism enables lagosin to be produced in good yield and in an economic manner.

Since the structure of lagosin is at present undetermined it is first necessary to describe its characteristics. Some characteristics are described in the literature above referred to but for completeness we now set out the full characteristics we have observed.

THE CHARACTERlSTlCS AND PROPERTIES OF LAGOSlN Lagosin is a very pale yellow in colour and crystallizes in soft needles from methanol, M.P. (uncorrected), 230- 240 C. (deo). The substance has a specic rotation of [o1]D2=-160 (c., 0.2 in MeOl-l). It gives a bluishvioiet colour with cold, concentrated sulphuric acid, which colour stable for 24 hours. Lagosin is sparingly soluble in water, slightly soluble in methanol, ethanol, butanol; readily soluble in dimethylformamide, glacial acetic acid and pyridine. lt is insoluble in chloroform trichloroethylene, ether and petroleum ethers.

Microanalysis of lagosin gives C, 61.25%; H, 9.12%; N, nil. On hydrogenation lagosin gives a derivative, M.P. 156-7", [o1]D2-{-3.5 (c., 1.98 in MeOH), crystallographic determination of molecular weight, 810i15, which indicates the empirical formula for lagosin to be C41H66 70O14 and for the hydrogenation product, C41`78 80014. CME-7168014 requires C, H,

We have established a standard of activity for lagosin by assigning to an early partially pure isolate an activity of 1000 units/mgm. Crystalline lagosin in the purest form obtained by us has an activity on this scale of 4950 units/mgm. Saccharomyces cerevisiae NCTC4614 was use-d as standard test organism and assays are conducted by the agar cup plate method.

A solution of lagosin in methanol exhibits an ultraviolet absorption spectrum similar to the polyene antibiotics lipin (Whitield, Brock, Ammann, Gottlieb and Carter, l. Amer. Chem. Soc. (1955), 77, 4799) and fungichromin (Tytell, McCarthy, Fisher, Bolhofer and 3,013,947 Patented Dec. 19, 1961 Charney, Antibiotics Annual, 1954-55, p. 716), with three characteristic maxima of El@ 1475 at 357 m11 1490 at 339 m11, and 927 at 323 m11; a shoulder appears at 308 m11.

A nujol-mull of lagosin gives a characteristic infra-red absorption spectrum, shown in the accompanying drawing, which is similar to that given by lipin and by fungichromin; strong absorption bands are shown at 3580 (2.7911), 3360 (2,9811), 2900 (3.4511), 2840 (3.5211), 1716 (5.83/1), 1462 (6.8411), 1374 (7.2811), 1138 (8.7911), 1040 (9.6111), 1008 (9.9311) and 846 cin-1 (11.8211), medium absorption bands at 1340 (7.4611), 1326 (7.5411), 1178 (8.4811), 1088 (9.1911), 1068 (9.36/1), and 722 cm."1 (13.8511), and weak absorption bands at 1645 (6.0811), 1238 (8.0811),1110 (9.01/1), 975 (10.2611), 896 (11.1611), 805 (12.4211) and 764 ein#1 (13.09/1). Lagosin is differentiated from tilipin by the fact that the bands at 1178 (8.4811), 1088 (9.1911) and 1040 cm.1 (9.61/1) are about 25% weaker and the band at 1068 cm.-l (9.36/1) about 50% stronger than the corresponding bands in the ilipin spectrum. it is differentiated from fungichromin by the absence of the band at 1587 cmi-1 (6.3011) shown by fungichromin and by possessing a band at 132-6 cm.-1 (7.5411) not shown by fungichromin.

Lagosin appears to be distinguished from fungichromin and lipin by a number of characteristic differences. Thus, lagosin has an empirical formula of C41H66 70O14 whereas fungichromin has an empirical formula of C35i-160013 and filipin an empirical formula of C30H50O10. The melting points are different, lagosin having an M.P. of ZBO-240 C., fungichromin and M.P. of 20S-210 C. and tilipn an M.P. of 195-205 C. ln the U.V. the ratios of the El?, maxima at 357mg. and 339m# of lagosin and fungichromin are different; for lagosin the ratio is 1:1 whereas for fungichromin 1.1:1. Filipin is converted to a colourless degradation product by methanol at 4 C.; no such change occurs with lagosin.

Lagosin, both as a solid and in solution is sensitive to light. The solid becomes a deeper yellow and nally a pale buff colour. Solutions (50 11g/ml. in ethanol) showed no loss when stored in the dark at room temperature for five weeks.

The microbiological spectrum of lagosin was determined by reference to the minimal concentration required completely to inhibit the growth of certain phytopatho` genio fungi. Partly purified material assaying at 1760 u./mgm. dissolved in methanol and diluted with water to give a methanol concentration of 0.060.25%, was used. The results were as follows:

Type of Minimal Species Inoculum: Inhibitory M=Mycelial, Concentration S= Spor-es in Units/ml.

Altemmia salami- Botti/tis alZiz'. 17. 5 Botryts fabae--.. 17. 5 C'Zadospon'um f 111mm. 70 Clastcroiporium tu., pu, L 70 Corticium salam' 17.5 Diplo/lia f "is 70 Elsinoe ampelina 35 Fusarium coerulf'z17n 70 Fusarium culmurum 17. 5 Fusarium m'vale 35 Glomerella cngulata 70 Do 35 Sclerotma Camellia- 70 Stereum purpareum 70 Verticillum aZbo-atrum.. 70

Lagosin is active against a range of fungi and yeasts at low concentrations as is shown in the following table:

Minimum Inhibitory Cono., {rg/Inl Species The toxicity of lagosin to mammals on oral administration is low, the LD50 being 2100 mg./kg. body weight for mice and 1750 mg./kg. for rats. Lagosin shows a substantially complete absence of phytotoxicity, whether applied to roots or foliage of, for example, the rose, tomato, dwarf bean and antirrhinum.

The antibiotic lagosin has a wide field of application as it is generally useful in inhibiting or controlling the growth of pathogenic and undesirable yeasts and fungi.

THE CHARACTERISTICS OF S T REPT OM YCES ROSEO-LUTE US When first isolated from soil, Streptomyces roseo-Intens was grown on malt agar plates and sections of the colonies transferred to plates seeded with various fungi. Zones of inhibition were observed in Candida albicans, Saccharomyces cerevisiae, Aspergillus niger and T richoplzyton mentagroplzytes.

Streptornyces roseo-Intens grows in the form of a muchbranched mycleium with characteristic aerial mycelium and conidiospores formed in chains. It is aerobic and mesophilic. Its growth characteristics vary somewhat depending on the medium on which it is grown. The growth characteristics on the media mentioned below are given by way of example only:

Asparagine dextrose zgan- Good growth; vegetative mycelium creamy white; profuse aerial mycelium white becoming light vinaceous fawn; the reverse of the colony empire yellow deepening to yellow chrome with age; a bright yellow pigment diffused through the agar.

Yeast extract agata- Vegetative mycelium orange becoming rufous or apricot orange with age; aerial mycelium white and at first scant; a soluble golden pigment diffused through the agar.

Bennetts zgan-Aerial mycelium abundant, white becoming pinkish grey; reverse colony light cadmium; a yellow pigment diffused through the agar.

Casein agar.-Aerial mycelium abundant, white becoming pinkish grey, then yellow and finally at 24 days a greenish grey; the reverse of the colony was bright orange-chrome and a golden pigment diffused from the colony; the casein around the colony was cleared.

Calcium malate agar.--Very poor growth; colonies smooth and cream coloured; some yellow pigment diffused through the agar.

Tyrosine agar.-Vegetative mycelium primuline yellow in colour; aerial mycelium sparse and white; no decomposition of tyrosine at 24 days.

Cellulose again-No appreciable growth at 24 days.

Stare/z again- Sparse cottony growth even at 24 days; aerial mycelium white becoming vinaceous buff.

Czapek Dox again-Growth sparse; initially cottony and white, becoming vinaceous fawn.

Synthetic agar with dextrose-Good growth; aerial mycelium brownish pink; a bright yellow pigment diffused through the agar.

Lizmus miIk.-Peptonised with an acid reaction; heavy growth at surface covering all but centre of tube.

Potato plug- Vegetative mycelium ochraceous orange becoming cinnamon brown; aerial mycelium maize to baryta yellow; a soluble yellow pigment diffused through the plug.

Gelatin stab- Discrete spherical colonies grew in the stab; liquefaction only after 5 Weeks incubation.

Nitrate broth.-No nitrite formed; nitrate still present after 24 days.

Glucose nutrient broth-Thin ring of white growth at the surface and iloccular growth at the bottom of the tube.

All colour descriptions referred to above conform to Ridgeway, Colour standards and Nomenclature, Washington, 1912.

The morphology of Streptomyces roseo-luteus was studied by growing it on cellophane discs on Bennetts agar and on asparagine-dextrose agar, the organism being examined after 10 days incubation. On Bennetts agar the spores were elliptical and borne in long closely spiralled chains, whilst on asparagine-dextrose agar the spores were oval to elliptical and borne in long chains loosely spiralled. The average spore size ranges from about 1.3-1.7a with an average size of about l.5,a.

The ability of Streptomyces roseo-Intens to utilise various carbon compounds was tested under the conditions described by Pridham, T. G. and Gottlieb D. (I. Bact., 1948, 56 (l), 113).

The following compounds were readily utilised:

Arabinose, salicin, raffinose, mannitol, scrbitol, xylose, trehalose, lactose, maltose, melibiose and dextrose.

The following compounds were not utilised and supported very sparse growth:

Erythritol, adonitol, aesculin, rhamnose, inulin, sucrose, sodium citrate, sodium acetate, fructose, dextran, sorbose and melezitose.

Streptomyces roseo-linens is morphologically distinguished from other Streptomyces of which we are at present aware, inter alia, by the following characteristics, namely, the vinaceous fawn colour of the aerial mycelium and the production of a bright yellow soluble pigment on both organic and synthetic media, the peptonisation of mill: and the very slow liquefaction of gelatine.

Following Bergeys Manual of Determinative Bacteriology Streptomyces roseo-Intens appears to fall in Group IASa2 or IASa3 of that manual. By comparison Streptomyces penticus appears to be characterised by the production of a soluble brown pigment in organic media and thus falls in the broad group Ib of the Bergey Classification; Streptomyces penticus is also said to be very similar to Streptomyces rubireticzrli, which also falls into group Ib of Bergey and from which Szreptomyces roseo-linens is markedly different.

THE CULTIVATION OF ST REPT OM YCES ROSEO- LUT EUS TO YIELD CRUDE LAGOSIN Lagosin is produced by the aerobic culture of Streptomyces roseo-Intens in or on a medium capable of supporting the growth thereof. Suitable media are those generally used for the culture of moulds of the genus Streptomyces, and should basically contain one or more assimilable sources of nitrogen and an assimilable source of carbon and energy, and nutrient salts. The culture is preferably conducted under submerged aerobic conditions.

It will be understood by those skilled in the art that the organism Streptomyces roseo-Intens is capable of mutation to mutant strains also capable of yielding lagosin. It is thus the practice in the antibiotic industry to produce mutant strains of antibiotic-producing organisms for the purpose of obtaining improved yields and/r more economic production. The present invention thus includes within its scope the use of lagosinproducing mutants of Streptomyces roseo-Intens for the production of the antibiotic.

The source of nitrogen is preferably in the form of complex organic material such as oatmeal, peptone, soya meal, maize meal, corn steep liquor, meat extract or a casein digest. Frequently such complex sources of nitrogen will be found to contain also the nutrient salts required by the organism. The source of carbon and energy may for example be carbohydrate assimilable by the mould, for example, glucose, lactose, starch, or assimilable oils or fats eg. palm oil, maize oil or lard oil; glycerol and palmitic acid are also suitable. Such compounds may be already present in the nitrogen source or may be added to the culture media separately.

It should be noted that the presence of palm oil, maize oil or lard oil or palmitic acid to the culture media results in considerable increase in the yield of lagosin, the yield being some two to four times greater than that achieved using the same medium without one of these constituents. The palm oil, maize oil, lard oil or palrnitic acid is preferably added in an amount of from 2.5-7.5%, advantageously approximately Examples of suitable media for use in the process of this invention are the following:

Oatmeal medium Soya medium Oatmeal 25 Soya flour 30 Chalk 20 Distillers solubles-. 7. 5 Dextrosel 5 Sodium chloride 2. 5 Dist. Water to 1l. pH 6.8:l:0.1. Dist. water to 1 l. pH

Potato-meat medium Glycerol-meat medium Tryptone, g 5 Glycerol, cc 20.0 Soluble starch, g Pantone, g l0 Difco beef extract, g 3 Sodium chloride, g 10 Dextrose, g.-- 2 Lab Lernco, g 5 KgHPOr, g. 1. 2 KzHPOi, g 5 KHZPO 0. 8 Ferrous sulphate (0 a Distilled water to 1l.

ous solution), ml 1.0 Potato extract to 11. pl 6.9:l:0.1. pH 6.8:l;0.1.

The following two media have been found to be particularly satisfactory for large-scale production.

Medium 1: Percent Oatflour 3 Whey powder 3 Chalk 1 Glucose 0.75

(pH adjusted to 6.9-7.0 with sodium hydroxide; sterilized 45 min. at 120 C.)

Medium 2: Percent Oatour 3 Whey powder 3 Palm oil 5 Chalk 1 Glucose 0.75

It will be clear to those skilled in the art that media, other than those mentioned above can be used, and Whether any medium will support the growth of Streptomyces roseo-Intens for the production of lagosin can readily be determined by trial fermentation.

In order to produce lagosin on a large scale, spores of Strepmmyces roseso-luzetzs (which can be preserved by lyophilisation in dextran or soil) can, for example be transferred to a suitable glycerol-meat medium of the type detailed below (40 ml. per 250 ml. flask or 500 ml. per 2 l. ask) and incubated for 48 hours on a rotary shaker at 29 C. In this way a heavy mycelial growth is developed.

This vegetative inoculum is then used to inoculate shake flasks or stirred vessels containing a development medium, for example:

Percent Glycerol K 5 Meat extract 1 Casein digest 1 Sodium chloride 0.5 (pH 6.8-7.0; sterilized 30 min. at 120 C.)

Percent Glucose l Meat extract 0.3 Casein digest 1 (pH 6.8-7.0; sterilized 30 min. at 120 C.)

THE ISOLATION AND PURIFICATION OF LAGOSIN Lagosin is contained in both the mycelium and in the culture broth, a far larger proportion of the total lagosin present being in the mycelium. It can be extracted from either the culture broth or the mycelium by means of a suitable organic solvent, for example butanol, amyl al.- cohol, ethyl acetate and butyl acetate, of which butanol is preferred. On the addition of ether to the butanolic extract, after it has been evaporated to a small volume, crude lagosin precipitates, and can be ltered off. After washing in ether it can be dried in vacuo, to give an orange-yellow solid which, in general, assays between 1,000 and 2,000 u./mgm.

Further purification of the product can be effected in lany convenient manner. Thus, the crude material may be subjected to a further washing with ether, and then washed with petroleum ether and dissolved in warm methanol. Evaporation to about one-third volume and chilling gives a pale yellow precipitate assaying at `about: 3,000 u./mgm.

In an alternative method the crude material may be Washed repeatedly with water until no further colour is removed. After drying, the product is dissolved in warm methanol, concentrated to about three-quarters volume and then allo-wed to stand overnight. Pale yellow solid having a potency of about 3500 u./mgm. is deposited.

In a preferred method of operation the broth is filtered on a kieselguhr pre-coated filter and the mycelial solid extracted twice with n-butanol. The combined butanol extracts are then evaporated in vacuo to a small volumel and then, by the addition of water and continued evaporation, the butanol entirely replaced by water. The crude solid is collected by centrifugation, washed with ether and dried. This material is extracted in a soxhlet with methanol, the purified lagosin separating from the extract on standing as a pale yellow semi-crystalline mass assaying about 4000 u./mgm. On recrystallisation from methanol the pure compound is obtained in cushions of soft, very pale yellow needles, assaying about 4950 u./mgm.

For the better understanding of the invention the following examples are given by way of illustration only:

Example l Percent Glycerol 5.0 Meat extract 1.0 Casein digest 1.0 Sodium chloride 0.5

pH adjusted to 5.8-7.0. (Sterilised for 30 minutes at This was stirred at 350 revolutions per minute (rpm.) aerated at 8 cubic feet per minute (cfm.) and maintained at a temperature of 28 C. for 24 hours, at the end of which time there was prolific growth.

45 litres of this well grown culture were transferred to 450 litres of sterile production medium:

VPercent Oattlour 3.0 Light precipitated chalk 1.0 Whey powder- 3.0 Glucose 0.75 -pH Vadjusted to 6.9-7.0 with sodium hydroxide. (Sterilized for 30 minutes at 120 C.)

This was stirred at 350 r.p.m., aerated at 20 c.f.m. and maintained at a temperature of 28 C. for 112 hours. At this time the broth assayed at 4275 u./ml.

2% (w./v.) of kieselguhr was added to 425 litres of the broth containing an estimated 1930 m.u. of lagosn. The mixture was filtered on a ro-tary filter using a kieselguhr precoat giving 58 kg. of cuttings and 350 litres of filtrate containing an estimated 105 m.u. of lagosin (by assay).

The cuttings were eluted with butanol, first with 100 litres from which 70 litres were recovered by decantation, secondly with 70 litres from which 60 litres were recovered, thirdly with 50 litres from which 40 litres were recovered and nally with 50 litres from which all the solvent was recovered. The total quantity of lagosin in the combined butanol eluates was 1040 m.u. (i.e. 53.8% of the original total by assay).

The combined eluates were concentrated under vacuum to approximately 6 litres and then water added so as to replace the evaporating butanol. The aqueous suspension was mixed with ether to remove the red oil and filtered. The solid was then re-washed with ether completely to remove a red oil which was present. The solid was then extracted in a soxhlet with methanol, the lagosin which separated being removed from the ask at periodic intervals to give a total of 247 g. of lagosin approximately 75% pure.

To remove impurities, 140 g. of this material, approximately 75% pure was extracted in a soxhlet for 2 hours with chloroform, air dried, then extracted for 2 hours with petroleum ether, air dried, and then for 2 hours with ethyl ether. The solid was finally air dried.

This solid was re-extracted in a soxhlet with methanol 8 for 8 hours. On cooling the methanol overnight 5.75 g. of pure lagosin were obtained. By reiluxing the solid remaining in the soxhlet thmble for 1 hour with the mother liquor from above, filtering and allowing to stand overnight, a further 25 g. were obtained.

Example 2 The beneficial effect on the production of lagosin by the addition of palm oil, lard oil or palmitic acid was shown by the following experiment:

Twelve fermenters each containing three litres of production medium as described in Example 1 were set up. To 4three was added 5% palm oil, to another three 5% lard oil and to another three 5% palmitic acid. The remaining three had no addition and served as controls The following results were obtained, each figure being the average titre, by microbiological assay, of the three fermenters:

"Controls Plus 5% Plus 5% Plus 5% Hours (no addi- Palm oil, Lard otl, Ialmtic tion) u./ml. u./rul 11./m1. acid, u./ml.

Example 3 In another experiment maize oil and oleic acid were compared with palm oil and palmitic acid. The following results were obtained which show that maize oil is of value in obtaining good yields of lagosin but oleic acid is of less value.

Oil added Maximum titre, u./ml.

5% Palm Oil 31, 7' 5% Masc Oil 21, 300 5% Palmitlc Acld.- 21, 860 5% Oleic Acid 865 Example 4 Lagosin may also be extracted without the prior filtration of the mycelium from the broth.

A medium consisting of 4% oatmeal and 5% maize oil was fermented as described in Example l. Without ltration, litres of the whole broth, containing 373 g. of lagosin, were mixed with a half-volume of n-butanol and the mixture separated centrifugally on two Laval separators in series. The butanolic extracts were evaporated under reduced pressure below 45 C. to about onefifth and then two volumes of methanol were added with stirring. Unfermented maize oil separated at this stage and was removed by centrifugation. The methanol-bu tanol solution was clarified by ltration through kieselguhr and then evaporated until the methanol was removed leaving a supersaturated butanolic solution of lagosin. The solution was cooled rapidly with vigorous stirring and allowed to stand at room temperature for an hour. The lagosin was filtered, washed with methanol and then with ether (removing a red oil) and the material dried under vacuum, yielding 190 g. of product.

This application is a continuation-impart of application Serial No. 815,849 filed May 26, 1959, by Christine Joy Bessel] et al., now abandoned.

We claim:

1. A process for the production of lagosin which comprises cultivating an organism selected from the group consisting of Streptomyces rOseo-Iuleus (NRRL 2776, NCIB 8984) and a lagosin-producing mutant thereof in a nutrient medium therefor and recovering the lagosin from the medium.

2. A process as claimed in claim 1 in which the culture is conducted under submerged aerobic conditions.

3. A process as claimed in claim 2 in which the medium contains, as a source of assimilable nitrogen, at least one material selected from the group consisting of oatmeal, peptone, soya meal, maize meal, meat extract and casein digest.

4. A process as claimed in claim 2 in which the medium contains, as a source of carbon and energy, at least one material selected from the group consisting of glucose, lactose, starch, palm oil, maize oil, lard oil, glycerol and palmitic acid.

5. A process as claimed in claim 4 in which the medium contains palm oil in an amount of from 2.5-7.5

6. A process as claimed in claim 4 in which the n1edium contains maize oil in an amount of from 2 5-7.5%.

7. A process as claimed in claim 4 in which the rnedium contains lard oil in an amount of from 2.5-7.5%.

8. A process as claimed in claim 4 in which the rnedium contains palmitic acid in an amount of from 2.57.5%.

9. A process as claimed in claim 2 in which the lagosin is recovered by extracting the lagosin-contaning material 20 with an organic solvent for lagosin and recovering the lagosin from the organic solvent.

10. A process as claimed in claim 9 in which the ori ganic solvent is selected from the group consisting of butanol, amyl alcohol, ethyl acetate and butyl acetate.

1l. A process as claimed in claim 9 in which the lagosin is recovered from the organic solvent extract by evaporation in vacuo with simultaneous replacement of the solvent Water.

12. A process as claimed in claim 9 in' which the lagosin is recovered from the organic solvent solution by addition of ether thereto, the precipitated lagosin being redissolved in methanol and allowed to crystallize.

References Cited in the le of this patent UNlTED STATES PATENTS 2,538,721 Colingsworth lan. 16, 1951 2,779,705 Peterson et al Jan. 29, 1957 FOREIGN PATENTS 762,702 Great Britain Dec. 5, 1956 OTHER REFERENCES Journal of Antibiotics, Series A, 1958, pages 26 to 29. 

1. A PROCESS FOR THE PRODUCTION OF LAGOSIN WHICH COMPRISES CULTIVATING AN ORGANISM SELECTED FROM THE GROUP CONSISTING OF STREPTOMYCTES RESOE-LUTEUS (NRRL 2776, NCIB 8984) AND A LAGOSIN-PRODUCING MUTANT THEREOF IN A NUTRIENT MEDIUM THEREFOR AND RECOVERING THE LAGOSIN FROM THE MEDIUM. 